Src homology 2 (SH2) domains selectively bind to phosphotyrosyl (pTyr)-containing regions of target proteins. SH2 binding can result in the modulation of c-src activity (Cooper et al., Cell 73: 1051-1054 (1993)), the alteration of the substrate specificity of c-abl proto-oncoproteins (Mayer et al., Mol. Cell. Biol. 14: 2883-2894 (1994); Feller et al., Trends Biochem. Sci. 19: 453-458 (1994)) and the transduction of signals initiated at growth factor receptors (Margolis et al., J. Am. Soc. Nephrol. 5: 1288-1299 (1994)) and cellular attachment systems (Schlaepfer et al., Nature 372: 786-791 (1994)). Thus, the SH2 domain has been suggested to be a promising site for therapeutic intervention (Brugge, Science 260: 918-919 (1993)).
The SH2 domain of growth factor receptor-bound protein 2 (Grb2), a 24 kda intracellular carrier agent that is composed of one SH2 domain and two src homology 3 (SH3) domains, mediates cellular signaling by binding pTyr-containing motifs within several proteins, although the Grb2 SH2 domain also can bind to proteins that do not contain pTyr (Oligino et al., J. Biol. Chem. 272: 29046-29052 (1997)). Such proteins include the adapter protein SHC (via p52 binding; Pelicci et al., Cell 70: 93-104 (1992); Rozakis-Adcock et al., Nature 360: 689-692 (1992); Sastry et al., Oncogene 11: 1107-1112 (1995); Janes et al., Oncogene 9: 3601-3608 (1994); Daly et al., Oncogene 9: 2723-2727 (1994)), growth factor receptors, such as epidermal growth factor receptors (EGFR) and their oncogenic analog receptors, the erbB receptors (Rozakis-Adcock et al. (1992), supra; Lowenstein et al., Cell 70: 431-442 (1992); Gale et al., Nature 363: 88-92 (1993); Buday et al., Cell 73: 611-620 (1993); Egan et al., Nature 363: 45-51 (1993)), in particular erbB2 (HER-2 or neu; via p185 binding; Sastry et al. (1995), supra; Janes et al. (1994), supra; Daly et al. (1994), supra; Xie et al., J. Biol. Chem. 270: 30717-30724 (1995); Gishizky et al., PNAS USA 92: 10889-10893 (1995)), morphology-determining proteins, such as cytoplasmic focal adhesion protein-tyrosine kinase (FAK; Schlaepfer et al. (1994), supra), and cellular oncoproteins, such as BCR-abl (Gale et al. (1993), supra; Pendergast et al., Cell 75: 175-185 (1993); Xie et al. (1995), supra; Gishizky et al. (1995), supra).
The prevention of Grb2-mediated multiprotein assemblies is considered to be a promising therapeutic target for the development of antiproliferative agents directed to cells that over-express growth factor receptors. In this regard, a non-phosphorylated peptide containing 11 amino acids in which the side chains of the two terminal cysteine residues form a ring has been reported to bind selectively the SH2 domain of Grb2 (Oligino et al. (1997), supra). The cyclic structure and the size of the ring have been determined to be critical to the binding activity of the non-phosphorylated peptide, which has been designated G1. An equipotent redox-stable thioether cyclized analog, G1TE, also has been reported (Oligino et al. (1997), supra). Unfortunately, G1 and G1TE have receptor affinities that are substantially lower than a segment of the naturally phosphorylated ligand, i.e., SHC(pY317)-9-mer (IC50 of 10-25 μM and 10-15 μM, respectively, vs. 1.0 μM). Accordingly, the utility of G1 and G1TE in in vivo prophylactic and therapeutic applications is limited.
In view of the above, there remains a need for an effective inhibitor of binding of an SH2 domain in a protein comprising an SH2 domain to a target protein. Therefore, it is an object of the present invention to provide non-phosphorylated cyclic peptide inhibitors of SH2 domains to target proteins, such as the SH2 domain that exists in Grb2 protein, which are redox-stable in vivo and are characterized by receptor affinities that are substantially better than currently available cyclic peptide inhibitors of SH2 domains. It is another object of the present invention to provide conjugates of such cyclic peptide inhibitors in order to facilitate cellular internalization of active agents. It is yet another object of the present invention to provide a composition comprising (i) such an inhibitor or such a conjugate and (ii) a carrier. It is still yet another object of the present invention to provide a method of synthesizing such conjugates. A further object of the present invention is to provide methods of use of such inhibitors and conjugates thereof. These and other objects and advantages, as well as additional inventive features, will be apparent from the detailed description provided herein.